WO1999003517A1

WO1999003517A1 - Implantable device coated with polymer capable of releasing biologically active substances

Info

Publication number: WO1999003517A1
Application number: PCT/FR1998/001538
Authority: WO
Inventors: Jean Luc Dubois-Rande; Trung Le Doan; Minh Chau Pham; Benoît PIRO; Emmanuel Teiger; Jean Pierre Tenu
Original assignee: Centre National De La Recherche Scientifique
Priority date: 1997-07-16
Filing date: 1998-07-15
Publication date: 1999-01-28
Also published as: FR2766092A1; FR2766092B1; ES2212328T3; EP1001818B1; DE69820268D1; EP1001818A1; DE69820268T2; US6468304B1

Abstract

The invention concerns the field of devices implantable in the system. The device comprises an electroconductive support coated with an electroconductive polymer deposit derived from pyrrole, naphthalene or thiopene, whereon is encapsulated at least one biologically active substance of anionic or cationic type, and in particular an oligonucleotide antisense. The polymer deposition on the support is carried out, in a first step, by electropolymerisation directly on the metal support, or in another embodiment, by deposition of the polymer in a solution on the metal support; then, in a second step, by oxidation or electrochemical reduction and fixing of the biologically active substance of anionic or cationic type on the polymer. The invention is applicable in cardiology for preparing stents for treating restenosis.

Description

IMPLANTABLE DEVICE COATED WITH A POLYMER CAPABLE OF RELEASING BIOLOGICALLY ACTIVE SUBSTANCES

The present invention relates to an implant coated with a polymer capable of releasing various substances, and more particularly a stent coated with a polymer capable of encapsulating biologically active substances intended for the treatment of restenosis, and of releasing them locally, as well as 'a process for depositing a polymer on a support.

In various fields of medicine, techniques have been developed which consist in introducing implants or prostheses into the human or animal body in order to correct or limit certain deficiencies.

Thus, in the field of cardiology, the technique of balloon angioplasty of arterial stenoses is today widely used for the treatment of coronary or peripheral vascular lesions responsible for angina pectoris, myocardial infarction and arteritis of the lower limbs. This technique consists in introducing, under angiographic control, an inflatable balloon probe into the vessel to be treated at the level of the narrowed area, and in restoring the blood flow by crushing the atheroma plate in the wall of the vessel. It is thus generally possible to avoid resorting to conventional surgical techniques to achieve arterial or peripheral revascularization.

However, angioplasty has the disadvantage of often causing further narrowing of the artery, called restenosis. In about 30% to 40% of cases, this restenosis occurs within six months, and is unpredictable. It is mainly due to a retractable scarring of artery and a proliferation of smooth muscle cells of the arterial wall, in response to the aggression produced by the introduction of the balloon. These two phenomena lead to a further narrowing of the arterial lumen. The administration of drugs such as angiotensin converting enzyme inhibitors, anti-inflammatories, anti-platelet aggregants, anticoagulants, etc., has not been able to effectively combat the phenomenon of restenosis . To prevent arterial retraction, metallic endovascular prostheses in the form of a small spring, called stents, are used and placed in the artery after dilation. The effect of the stent is to prevent immediate elastic return of the artery wall, and, in the longer term, to prevent constriction of the artery. On the other hand, the presence of the stent does not have a favorable effect on the proliferation of smooth muscle cells, and in certain cases it can even worsen it.

It is therefore desirable to be able to combine an effective drug treatment with the placement of the stent.

It is known that certain antisense oligonucleotides exert antiproliferative activity in vitro and in vivo (MR Bennett et al. "Inhibition of vascular muscle cell accumulation in vitro and in vivo by c-myc antisense oligonucleotides", .T. Clin. Invest. (1994) 93, 820-28). However, no effective technique for administering such compounds has been described. Antisense nucleotide derivatives adsorbed or encapsulated in alkyl polycyanoacrylate nanoparticles are described in patent FR-A-2,724,935 which proposes this technique for their intra-tumoral administration in the treatment of certain cancers. Patent DE-A-4,429,380 describes a process for preparing stents with a ceramic or metallic support coated with an intermediate layer of amorphous silicon and with a layer of semiconductor material, these two layers being nested one in the other.

Metallic stents covered with a first composite layer of a polymer and a therapeutically active substance, coated with a second layer of fibrin, are described in patent application EP-A-701.802. The polymer used is chosen from silicones, polyurethanes, polyesters, polyethers and vinyl derivatives. On the other hand, according to patent EP-A-566,245, fibrin can be used in the treatment of restenosis. Tests carried out on pig coronary arteries with various biodegradable polymers (for example polyglycolic acid / poly-lactic acid, polycaprolactone, etc.) or non-biodegradable (polyurethane, silicone, polyethylene terephthalate) have shown significant inflammatory reactions accompanied of a fibrocellular proliferation of the arterial wall (WJ Van der Giessen et al., Circulation. (1996) 94, 1690-97).

Thus, there is currently no system effectively combining a stent and an antiproliferative substance of smooth muscle cells, for the treatment of restenosis.

The present invention therefore relates to a device implantable in the body, or endoprosthesis, and in particular a stent, covered with a polymer capable of encapsulating biologically active substances of anionic or cationic nature and of releasing them locally, usable in the treatment of various conditions, including endoprosthetic thrombosis as well as restenosis in the field of coronary angioplasty and angioplasty of the peripheral arteries.

The invention also relates to a method for depositing an electroconductive polymer containing a biologically active substance, on an electroconductive support such as a metal support, and in particular a stent.

The process according to the present invention allows the electrochemical deposition of an electroconductive polymer, for example a polymer derived from pyrrole, naphthalene or thiophene, which can be obtained in an aqueous medium under mild conditions. that is to say at ambient temperature and under moderate saline conditions starting from commercially available monomers. In addition, the electrochemical deposition allows precise control of the thickness of the polymer layer deposited on the support, and the medium used does not contain any chemical oxidant or organic solvent which could have harmful effects in the intended application. In the case where the electrochemical polymerization is difficult or cannot be envisaged, the polymer can be prepared in solution in an appropriate solvent, according to a conventional technique, and deposited on the support by spraying or soaking, then evaporation of the solvent.

The method of the invention essentially comprises two steps. In a first step, an electropolymerization is carried out directly on the metal support, or, according to a variant, the polymer is prepared in solution which is deposited on the metal support, as indicated above. Then, in a second step, an oxidation (if the polymer is cationic) or a reduction (if the polymer is anionic) is carried out, and simultaneously the substance is fixed biologically active on the polymer. Oxidation and reduction, as the case may be, are carried out electrochemically, in a known manner by creating a potential difference between the electroconductive support and the polymer in order to form positive or negative charges, respectively, favoring the fixing of the active substance. For example, the formation of a polymer matrix promotes the fixation of an active substance constituted by a positively charged phosphorylated nucleic base; conversely, the formation of positive charges in the polymer promotes the attachment of an active substance constituted for example by an oligonucleotide or a protein carrying negative sites such as ATP, or more generally any negatively charged particle, such as l heparin, a plasmid gene vector, or a linear or circular DNA fragment of the plasmid type.

According to a preferred form of implementation of the electroconductive polymer deposition process in accordance with the invention, the electrochemical polymerization is carried out in the presence of a water-soluble polymer, chosen from a polyethylene glycol, a polyvinylpyrrolidone, a polyethylene oxide, a copolymer of ethylene oxide and propylene oxide of the Poloxamer type, a polyethylene acetate, a polyvinyl alcohol, a polyacrylamide, as well as water-soluble derivatives of polyurethane. The presence of such a water-soluble polymer in the polymer matrix makes it possible to improve the permeability of the polymer to anionic species, such as oligonucleotides, used as active substance, and facilitates the controlled release of these molecules on contact with solutions containing competing ions, for example from the addition of sodium. Preferably, an amount of polymer representing between 4 and 10% of the total composition is used.

More particularly, the polymer film can advantageously be formed in two stages, the first consisting in polymerizing the monomer at a concentration of between approximately 0.01 and 0.1 M, to form a bonding layer of thickness less than approximately 1 μm, the second consisting in prolonging the polymerization reaction in the presence of water-soluble polymer in order to obtain a layer of thickness greater than 1 μm, which can be between 2 and 10 μm approximately.

It is advantageous to use a metal support produced for example from steel, a metal alloy, or a biocompatible metal and more particularly from stainless steel, tantalum, platinum, gold, nickel-titanium alloy or platinum-indium alloy. A stainless steel support can advantageously be used when the electrolytic medium does not contain chloride. It is also possible to use a non-metallic support such as a biologically compatible electroconductive charged polymer.

The oxidation is carried out, after deposition of the polymer as indicated above, by applying to the support an oxidation potential for a determined period of time. Thus, for example, depending on the polymer used, a potential of approximately 0.3 V to 1 V can be applied relative to a hydrogen reference electrode, for a period of time between 30 minutes and approximately 3 hours, in depending on the properties sought.

According to a characteristic of the invention, thanks to the oxidation step, it is possible to adjust the fixing capacity of the active substance, in particular the oligonucleotides. cleotides, on the polymer and then control the release locally on the site to be treated, once the stent is placed in the body. The loading of the active substance on the polymer is advantageously carried out simultaneously with the oxidation step, by adjusting the concentration of active substance in the oxidation solution to an appropriate value, which can be for example between 1 and 200 μM, depending on the substance used and the level of fixation sought. It is thus possible to achieve a binding rate greater than several nanomoles / mg of polymer, associated with slow release kinetics ensuring a prolonged effect of the active substance. This technique makes it possible, for example, to obtain release kinetics of the order of a few picomoles of active substance per mg of support and per day.

The release kinetics observed using the technique of the invention is related to the thickness of the polymer film. It is possible, in accordance with the invention, to adjust the kinetics of release of the active substance by choosing an appropriate thickness of the polymer on the support, for example between 2 and 10 μm approximately.

The active substance is chosen according to the use envisaged for the implant. Thus, in the case of a stent covered with a polymer intended for the treatment of post-angioplasty restenosis, the active substance is preferably an antisense oligonucleotide capable of selectively blocking the expression of genes controlling the proliferation of smooth muscle cells of the arterial wall. It is possible, for example, to use oligonucleotides, and more particularly antisense oligodesoxyribonucleotides (ODN) with a phosphodiester or phosphorothioate structure, directed against the receptor. insulin-like growth factor (or IGFR) as described by P. Delafontaine et al. ("Regulation of vascular smooth muscle cell Insulin-like Growth Factor I Receptors by phosphorothioate oligonucleotides" J. Biol. Chem, (1995) 270, 14383-388) and WO-A-96.10401, or oligo-deoxynucleotides containing n guanines contiguous, n can vary from 2 to 5 as described by Burgess et al. ("The antiproliferative activity of c-myb and c-myc antisense oligonucleotides in smooth muscle cells is caused by a non antisens mechanism", Proc. Nat. Acad. Sci. USA (1995) 92, 4051-4055).

These oligodesoxyribonucleotides generally have a very short lifetime in the free state in the organism because they are quickly digested by nucleases, which limits their effectiveness. The present invention is therefore particularly advantageous because it makes it possible to protect these oligodesoxyribonucleotides in the polymer matrix and to deliver them progressively to the site itself, where they can then act effectively. In addition, according to the invention, the oligonucleotide can be radioactively labeled. The radioactive isotope makes it possible to control the fixation on the polymer, and can also induce an antiproliferative effect on smooth muscle cells. The labeling can be carried out in a conventional manner, for example using sulfur S or phosphorus P, the half-life of which is 87 days and 15 days respectively, and is suitable for the development over time of restenosis.

The polymer used to cover the stent, in accordance with the present invention, is chosen from electrically conductive polymers having satisfactory properties of biological tolerance. A polypyrrole is preferably used, a poly (diaminonaphthalene) or a poly (3, 4-ethylene-dioxythiophene) or poly (EDT), which have suitable electrical conduction and mechanical strength properties, without harming the mechanical qualities of the support used. It may be advantageous, in accordance with the present invention, to apply to the polymer carrying the oligonucleotide, a layer of substance such as fibrin, known for its antithrombotic activity.

The invention applies very particularly to a stent covered with a polymer containing an active substance effective in the treatment of restenosis. It can be applied more generally to any implant capable of being covered with a cationic (or cationizable) or anionic (or anionizable) polymer layer which can serve as a reservoir of active substance carrying positive or negative charges, intended to be released. locally, for example substances having pharmacological properties useful in the treatment of leukemias, solid tumors and organ rejection. The examples below illustrate the invention in more detail without limiting its scope.

Example 1

Electropolymerization of 3,4-ethylenedioxythiophene is carried out using a conventional electrochemical cell with three electrodes: a platinum counter electrode, a silver wire covered with silver chloride serving as a reference electrode providing a constant potential, and a platinum working electrode sprayed onto a glass plate 0.6 cm in surface, giving a potential with respect to the counter electrode. The cell of the cell contains the monomer solution to be polymerized and the salt ensuring the electrical conduction.

The monomer is 3, 4-ethylenedioxythiophene to which a polyethylene glycol (3.10 M) or a polyvinylpyrrolidone (2.10 ^" M) is added. The electrolytic solution is PBS (phosphate buffered saline) at pH 7.4 containing the following salts : KCl (2.7 mM), NaCl (0.14 M), KH ₂ P0 ₄ (1.4 mM) and Na ₂ HP0 ₄ (6.4 mM).

The monomer is added to the PBS solution diluted 15 times. The potentiodynamic method (cyclic voltammetry) is used, the potential ranging from -0.1 V to +1.3 V relative to Ag / AgCl for 10 to 30 cycles. The oxidation of the monomer begins at around 0.9 V. The last cycle is stopped at the higher potential to obtain a homogeneous oxidized film. The film thickness, measured using a scanning electron microscope, is 2.5 μm (10 cycles) or 5.4 μm (20 cycles). The thickness of the film can be increased by increasing the number of cycles. However, beyond about 30 cycles, the mechanical properties of the film may be reduced. One of the oligonucleotides indicated below is then incorporated as active substance, by oxidizing the polymer at a potential of +0.7 V relative to a reference electrode Ag / AgCl, in a PBS solution containing an amount of oligonucleotide equal to 1 μM. The potential is maintained for approximately 1.5 hours.

The polymer film loaded with oligonucleotide is quickly rinsed with water on both sides of the working electrode.

The antisense oligodesoxyribonucleotides (ODN) used successively in this example are the following:

5 '-CTC-TCG-CAC-CCA-TCT-CTC-TCC-TTC-T (phosphorothioate) IGFR TCC-GGA-GCC-AGA-CTT-CAT-TC (phosphorothioate)

C-Myc 5 '-AAC-GTT-GAG-GGG-CAT (phosphodiester).

These ODNs are purified by high performance liquid chromatography (HPLC) on a C18 reverse phase column using tetraethylammonium acetate / acetonitrile elution buffers.

In order to control the encapsulation of the oligodesoxyribonucleotides (ODN) in the polymer film, the procedure is carried out by radioactive labeling according to a conventional technique, using P as an isotopic marker.

Labeling is carried out by transfer of a radioactive phosphate group ³² P from ATP to the 5 ′ position of the ODN, by the polynucleotide kinase at 37 ° C. in acetate buffer medium.

Example 2 was carried out one electropolymerization of a naphthalene derivative, 5-amino-l, 4-naphthoquinone (ANQ concentration 10 ^"2 M) in a solution of acetonitrile containing LiC10 ₄ IO ^{^} Η. We uses the potentiodynamic method (cyclic voltammetry) by varying the potential from 0.5 V to 1.45 V relative to a calomel reference electrode (ECS) for 40 minutes.

The film thickness, measured using a scanning electron microscope, is 1 μm.

The positively charged active substance is then incorporated by reducing the polymer to a potential of -0.3 V relative to an ECS in an aqueous solution of pH approximately 7 containing the positively charged substance. The potential is maintained for approximately 20 minutes. Example 3

The procedure is as in Example 1, using 3,4-ethylenedioxythiophene as the monomer, but forming the polymer film in two successive stages. In a first step, the monomer is polymerized in the same PBS medium as in Example 1, diluted 15 times. The monomer concentration is 3.10 ^" M. The technique used is identical to that of Example 1, but only carrying out 2 scanning cycles, so as to form a film about 0.5 μm thick, constituting a bonding layer.

In a second step, a polyvinylpyrrolidone with a molar mass of approximately 40,000, at a concentration of 2.10 ^" M, is added to the medium, and the polymerization is prolonged until a poly (3, 4-ethylenedioxythiophene) film having a thickness of 6 is obtained. approximately μm.

The tests carried out using a platinum stent as a support have demonstrated the excellent mechanical properties of the polymer coating which has good adhesion.

Example 4

The experiments carried out with the stents of the invention have demonstrated a very good tolerance after implantation in vivo, making it possible to envisage their use in the treatment of restenosis.

Thus, a platinum stent with a mesh structure, covered with polymer, prepared as indicated in Example 1, was implanted in the abdominal aorta of two New Zealand rabbits weighing 3.5 kg. An identical stent, but not covered with polymer, was implanted in the same animal, in the same aorta but 2 cm upstream and serves as a control stent. Treatment with aspirin was started one day before the intervention (0.07 mg / ml of drinking water), then continued for the duration of the implantation. The two arteries were removed, three and fifteen days after the placement of the stent, and examined in standard histology after fixation.

On the 3rd day, no parietal thrombus is observed. There is a moderate proliferation, not covering the stent meshes, of smooth muscle cells, at the contact areas between the stent and the arterial wall. We note the presence of some circulating cells, red blood cells and polymorphonuclear cells, as well as some collagen fibers. The appearance is identical at the level of the two stents, that is to say the control stent and the stent coated with polymer. On the 15th day, no parietal thrombus is observed. The proliferation of smooth muscle cells is a little more accentuated and consists of the same elements as above. The proliferative layer is more organized, covers the meshes of the polymer-coated stent and has a layer of endothelial cells on the surface, thus promoting the stabilization of the process.

These tests confirm the bio- and hemocompatibility properties of the poly (EDT) polymer used in accordance with the invention.

Claims

1. Device implantable in the body, characterized in that it comprises an electroconductive support covered with a layer of electroconductive polymer on which is encapsulated at least one biologically active substance of anionic or cationic nature.

2. Device according to claim 1, characterized in that the polymer is a polymer derived from pyrrole, naphthalene or thiophene.

3. Device according to claim 2, characterized in that the polymer is a poly (3, 4-ethylene-dioxythiophene).

4. Device according to any one of the preceding claims, characterized in that the biologically active substance is a phosphorylated nucleic base, an antisense oligonucleotide, or a vector of plasmid genes.

5. Device according to claim 4, characterized in that the biologically active substance is an antisense oligodeoxy-ribonucleotide (ODN) with a phosphodiester or phosphorothioate structure.

6. Device according to any one of the preceding claims, characterized in that the electroconductive support is a metal chosen from stainless steel, tantalum, platinum, gold, a nickel-titanium alloy or a platinum-indium alloy.

7. Device according to claim 1, characterized in that it is constituted by a stent.

8. A method of depositing a polymer on an electroconductive support for a device implantable in the body according to claim 1, characterized in that, in a first step, an electropolymerization is directly carried out. tion on the metal support, or, according to a variant, the polymer is prepared in solution which is deposited on the metal support, then, in a second step, an oxidation or reduction is carried out, and the biological substance is fixed. - Only active of an anionic or cationic nature on the polymer.

9. Method according to claim 8, characterized in that one electropolymerization is carried out in the presence of a water-soluble polymer.

10. Method according to claim 9, characterized in that the water-soluble polymer is chosen from a polyethylene glycol, a polyvinylpyrrolidone, a polyethylene oxide, a copolymer of ethylene oxide and of propylene oxide of the Poloxamer type, acetate of polyethylene, a polyvinyl alcohol, a polyacrylamide, as well as water-soluble derivatives of polyurethane.

11. Method according to claim 8, characterized in that the oxidation is carried out electrochemically.

12. Method according to any one of claims 8 to 11, characterized in that the loading of the active substance on the polymer is carried out simultaneously with the oxidation or reduction step.